Safety forms were approved on September 29, 2013 by the iGEM Safety Committee.

General

In a world where research in life science and especially in Genetic engineering and Synthetic biology is thriving, who is taking responsibility for all the genetically modified organisms that are being created and their potential harmfulness? Well, it is OURS of course! Every researcher should consider biosafety and bioethics as issues of high importance, before even starting to work with microorganisms. But first things first!

What is a Genetically Modified Microorganism (GMO)? What is Biosafety and Bioethics?

The [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf World Health Organisation (WHO)] defines:

• Genetically modified organisms (GMOs): as “organisms in which the genetic material (DNA) has been altered in a way that does not occur naturally” [http://www.who.int/foodsafety/publications/biotech/20questions/en/]. This means that the researcher removes or introduces genetic material to the organism. Additional material can either be of his own or somebody elses design or taken from other organisms. Every organism created in an iGEM project is therefore a GMO.

• Biosafety: is defined by the WHO as “the prevention of unintentional exposure to pathogens and toxins, or their accidental release, whereas biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins”. It is a very important part of lab work in general and work with GMOs in particular as these might pose a higher risk when released into the environment.

• Bioethics: is defined by the WHO as ‘’the study of the ethical and moral implications of biological discoveries, biomedical advances and their applications, as in the fields of genetic engineering and drug research.’’

General public is concerned when hearing about GMOs. The scientific community should spend more time communicating how useful GMOs are in everyday life, using simple examples to help people have a better understanding in what Synthetic Biology means and what can offer to the world.

In Denmark and in our university as well, biosafety is not an issue to be taken lightly. Everything is about researcher’s safety and all lab work is planned in respect to the environment. Local biosafety groups exist in every building (research group, department) and rules that apply to everyone are mandatory to be followed.

What is the regulation in Denmark: it follows the international guidelines of the [http://www.sns.dk/biosafety/english/ Cartagena Protocol] since 2002.

In DTU: We were working in two different laboratories, each in a different department: Systems Biology and the Environmental. Both labs have their own local biosafety group. Lab technicians and PhD students gave us a tour of the lab and provided us with all the information needed to work with the instruments in order to minimize the risk of accidents. It was requested from us to submit a Working Risk Assessment to show that we are aware of all the potential risks that our project held and to make sure that we knew what is considered to be a harmful reagent and how we would treat it. Also, to show that we are organizing our workspace according to the procedure that we want to perform (e.g. use a LAF bench to minimize risk of contamination), and that we always take care of our personal protection by wearing the necessary equipment (gloves, safety glasses when needed) and how to decontaminate biowaste and equipment used, especially in the case of spillage.

The department of Systems Biology has a biosafety board and specific safety rules applying to our department.

DTU has access to a Danish chemical portal called [http://www.kemibrug.dk/ Kemibrug], which provides safety information (safety data sheets) and other information for standard lab tasks. We were introduced to this database early in our project, from reading material and a lecture from Christopher Workman at our BioBrick Workshop in early March.

Prior to this, students working in the lab had relevant training from their bachelor studies and/or master courses.

All this combined, provided us with all the background (knowledge) required to treat all aspects of our project with responsibility and awareness of how important it is to make correct use of Synthetic Biology.

iGEM Safety Questions

Would any of your project ideas raise safety issues?

Researcher safety

• Microorganisms:

We are working with three different bacteria species.

The first one is E. Coli (K-12) and the strain we used is called TOP 10, is commonly used in a large number of labs. It belongs to Risk Group 1, it is not pathogenic, and also not able to survive a long time outside lab conditions. N. Europaea (ATCC 19718) is a common soil bacterium and does not pose a health risk. P. Aeruginosa PAO1 is a human pathogen with intrinsic resistance to antibiotics and disinfectants and also has environmental adaptability. However even though P. aeruginosa is capable of infecting healthy humans it is largely an opportunistic pathogen. The website of the Center for Disease Control states that “serious Pseudomonas infections usually occur in people in the hospital and/or with weakened immune systems. However, healthy people can also develop mild illnesses with P. aeruginosa, especially after exposure to water” [http://www.cdc.gov/hai/organisms/pseudomonas.html]. Gloves were worn at all times when handling P. aeruginosa cultures on plates or in liquid medium and special care was taken to not expose open wounds. Our final mutants do not incorporate genes from P. aeruginosa that make it pathogenic, so they do not have elevated risk over non-transformed E. coli.

• Reagents:

We are using the following reagents, which could potentially pose a risk.

[http://toxsci.oxfordjournals.org/content/59/1/5.full Nitric Oxide (NO)]: Should not be inhaled. Causes skin and eye irritation.

[http://www.sciencelab.com/msds.php?msdsId=9927667 Ethidium Bromide]: Should not be inhaled or ingested. Causes skin and eye irritation. Can be toxic when used often: mutagenic/carcinogenic.

[http://kemibrug.dk/KBA/CAS/104070/?show_KBA=1&portaldesign=1&lang=en Nitrous oxide (N₂O)]: Should not be inhaled, anaesthetic effect. Avoid skin contact.

[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=DK&language=EN-generic&productNumber=55459&brand=FLUKA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Ffluka%2F55459%3Flang%3Den Hydroxylamine (NH₂OH)]: Should not be ingested. Causes skin and eye irritation. Limited evidence of carcinogenic effects.

Public safety

There is only limited access to the lab (by authorized students and researchers) so the risk of release is very small. In case of release of Pseudomonas, it is considered to be a mild pathogen, and if an infection in humans was to occur, it can be treated with antibiotics. In addition to that P. aeruginosa is an abundant endogenous organism and has not been modified by our work in any way.

Environmental safety

Accidental release is very improbable because the strains we are working with are not able to survive outside lab conditions and biowaste material is treated with respect to the safety rules of our department. Equipment is either autoclaved or disinfected, and all students wash hands before leaving the lab.

The worst case risk would be if we chose to scale up the process, i.e. used our engineered E. Coli in wastewater treatment plants. If we do so, we will employ a suitable containment strategy. The E. coli strain that we are using will not be able to survive for long in non-regulated conditions. Also naturally occurring bacteria are routinely used in the treatment of waste water and the measures taken to sterilize the water before it leaves the treatment plant are suitable to neutralize our engineered bacteria as well since they do not posses increased resistance to sterilization methods.

Release of the produced nitrous oxide has to be strictly avoided since it has a number of adverse effects on the environment. Most importantly it is a strong greenhouse gas [http://www.reuters.com/article/2011/01/10/idUS213186050120110110].

Any nitric oxide produced during the denitrification process is assumed to be short lived since it will be converted rapidly into nitrous oxide. If however the conversion to nitrous oxide should fail, the nitric oxide needs to be contained as well, since it can be converted to nitric acid when entering the atmosphere, causing acid rains [http://nadp.sws.uiuc.edu/educ/acidrain.aspx]. Additionally there is also an abiotic conversion of nitric oxide into nitrous oxide, which as already mentioned is an ozone depleter.

Lastly the plasmids carrying the denitrification genes also hold antibiotic resistances in order to evoke selection pressure and avoid loss of them. It is therefore important that the engineered E. coli stay contained so that the resistance genes cannot be propagated by horizontal gene transfer.

Do any of the new BioBrick parts (or devices) that you made this year raise safety issues?

All our parts can be considered as biosafety level 1. The NirS region was taken from P. aeruginosa, which is itself BSL 2, however the part should not be classified as BSL 2 since it does not encode resistance or virulence genes. Our genes are under an arabinose inducible promoter, so they will not be expressed in the absence of arabinose, giving a small amount of control. After carefully evaluating potential risks, it was not a priority for us to add kill switches. However, if we were to scale this project up, we would do so.

Two of our BioBricks lead to the production of the potentially harmful gases nitric oxide and nitrous oxide. In huge quantities those gases pose risk to both human health and the environment. We therefore keep the transformants contained in bioreactors, which will minimize the threat as discussed above. In addition to that the amounts of gas produced during our project is small scale and if we were to take this project to a bigger scale, more severe methods for containment would be devised.

Is there a local biosafety group, committee, or review board at your institution?

We reviewed our project with the appropriate biosafety groups and wrote a Workplace Risk Assessment for our project. No concerns were raised during the review process. All lab work was performed in accordance with local and national guidelines. For details please see the General section above.

Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

We think that before starting the lab work, every team should plan their lab work according to safety requirements and perform a risk analysis. For instance, a workshop in order to introduce the biosafety guidelines would be helpful for future iGEM teams. A similar introduction by a lab technician who works in GMO labs, it is also beneficial. Lab protocols by standardized lab procedures and careful project design are really useful as well as a risk assessment. Then the worst scenarios would be taken in account. In order to avoid GMO release in the environment, kill switches could be used. A kill switch is a self-destruction mechanism which can work in genetic engineered genes, while they are released in the environment, then they can produce a toxic protein that damages bacterial cell membranes and kills the cells.